Transcription profiling of human cells expressing FoxM1-ER fusion protein

ABSTRACT: Experiment description To identify FoxM1-regulated genes, we carried out gene expression profile analysis after inducible activation of a FoxM1-ER fusion protein using high-density human cDNA microarrays. We constructed a stable line of U2OS cells expressing full-length HA-FoxM1 fused at its C-terminus to the ligand-binding domain of the estrogen receptor (ER), mutated such that it specifically responds to 4-hydroxytamoxifen (4-OHT) but not to estrogen. In the absence of 4-OHT the activity of the ER fusion protein is turned off, but can be rapidly induced when cells are exposed to 4-OHT. We isolated total RNA from untreated cultures and cultures treated with 4-OHT for 6 or 16 hr. As activation of FoxM1 results in accumulation of cells in G2/M, we additionally analyzed the induction of gene expression by FoxM1 in G1/S-synchronized cells by a thymidine block. Total RNA derived from untreated or stimulated FoxM1-ER cells was amplified, reverse transcribed, labeled and hybridized to cDNA microarrays containing 18,000 cDNAs and expressed sequence tags (ESTs). Bound cDNA was detected using Cy3 or Cy5 dyes. In each independent experiment we included reverted color controls (Cy3 (4-OHT treated); Cy5 (untreated) or Cy3 (untreated); Cy5 (treated)) and the expression profile was expressed as a Cy5:Cy3 ratio.

ORGANISM(S): Homo sapiens

SUBMITTER: Ron Kerkhoven 

PROVIDER: E-MEXP-230 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress